Ring - Substituted α ‐ Arylalanines for Probing Substituent E ff ects on 2 the Isomerization Reaction Catalyzed by an Aminomutase

7 ABSTRACT: β-Amino acids are emerging as an important class of 8 compounds in medicinal chemistry. β-Aryl-β-alanines show antiepilepto9 genesis activity, while others have been used to synthesize antibiotic β10 peptides. To assess the utility of a methylidene imidazolone-dependent 11 Pantoea agglomerans phenylalanine aminomutase (PaPAM) for making 12 non-natural β-amino acids, we surveyed the substrate specificity of 13 PaPAM with several commercially available (S)-arylalanine substrates. 14 Here, we report the Michaelis−Menten parameters and catalytic 15 efficiency of PaPAM for each substrate. Compared to phenylalanine, 16 substrates containing substituents that were either electron-withdrawing 17 or -donating through resonance or inductive effects affected the kcat of 18 PaPAM. Generally, the turnover and catalytic efficiency of PaPAM for 19 the meta-isomers were better than those for the corresponding paraand ortho-isomers, with some exceptions. PaPAM 20 principally synthesizes the β-amino acids at >90% and the cinnamate byproducts at <10% for 11 of the 19 productive substrates. 21 The yield from other substrates was 14−65% of the cinnamate analogue. Further, to explain the determinants of substrate 22 selectivity of PaPAM, a series of substrates with substituents on the aryl ring were docked into the crystal structure of the active 23 site. Induced fit of the protein to accommodate different substituents was modeled computationally by SLIDE docking. The 24 results provide insights into the roles of substrate orientation and conformational flexibility in turnover and indicate which terms 25 of the interaction energy should be accounted for the experimentally observed KM values, which largely determine catalytic 26 efficiency. Substrate selectivity of PaPAM is significantly influenced by steric barriers created by specific active-site residue 27 interactions with the substituted aryl portion of the substrate.